The overall goal of this proposal is to elucidate the scope of intracellular trafficking pathways responsible for the delivery of signaling and structura proteins into the light-sensitive organelle of photoreceptor cells, the outer segment. Almost everything we know about outer segment protein delivery relates to the visual pigment, rhodopsin, whereas the mechanisms responsible for the outer segment trafficking of other resident proteins remain largely unknown. During the next grant cycle, we propose to conduct detailed studies of two outer segment-resident proteins, peripherin and PRCD. Aim 1 will focus on the trafficking mechanism for the photoreceptor disc rim protein peripherin-2/RDS. We will employ live imaging, biochemical fractionation and quantitative mass spectrometry to elucidate whether peripherin and rhodopsin are packaged into the same or different transport vesicles and whether this packaging is performed by the same or different accessory proteins. Aim 2 will focus on PRCD, a novel constitutive component of photoreceptor discs whose mutations are associated with retinal degeneration in human patients and multiple breeds of dogs. Our proposed experiments will combine basic functional characterization of PRCD with investigation of its prevalent C2Y mutation that leads to PRCD mislocalization from the discs to other photoreceptor compartments. A particular emphasis will be placed on identifying PRCD interacting partners, which will benefit both of these directions. The proposed Aims are relevant to understanding the most basic issues in photoreceptor cell biology and are key for understanding the causes of many types of photoreceptor degeneration associated with defects in protein sorting, targeting and trafficking.